(a) Technical Field
The present invention relates to a ratiometric fluorescent probe which is selectively targeted to mitochondria, the representative active cellular organelles, and whose color of two-photon fluorescence changes upon binding to thiols, more particularly to a two-photon fluorescent probe capable of imaging the activity of thiols existing inside mitochondria with high sensitivity and selectivity by two-photon microscopy of low energy excitation source in real time and a method for preparing same.
(b) Background Art
Intracellular thiol derivatives (cysteine (Cys), homocysteine (Hcy) and glutathione (GSH)) play vital roles in biological activities. In particular, the thiol (RSH) derivatives in mitochondria play a key role in maintaining protein structure and redox balance through equilibrium with the disulfide (RSSR) forms. It is known that the ratio of thiol (RSH) to disulfide (RSSR) derivatives in the mitochondria is maintained at 100:1 or higher. A disruption of the balance may lead to severe cell damage and death.
To understand the roles of thiols, many one-photon fluorescent probes have been developed using fluorescein, rhodamine, green fluorescent protein (GFP), etc. as a fluorophore. However, most of these one-photon probes are problematic in that they require short excitation wavelengths (<500 nm), limiting their use in tissue imaging because of shallow penetration depth (<100 μm), photobleaching, cellular autofluorescence, and so forth. Two-photon microscopy (TPM) has been presented to solve these problems. Two-photon microscopy provides a number of advantages over one-photon microscopy, including increased penetration depth, localized excitation and prolonged observation time, because it utilizes two near-infrared photons of lower energy for excitation.
The probe market is increasing every year globally and about 4,000 one-photon fluorescent probes are commercially available. However, the number of two-photon probes developed thus far is only about 10, and, in particular, there is no two-photon fluorescent probe capable of selectively detecting thiols present in the mitochondria.